Thermo Fisher Scientific CarrierScan Assay 96-Array Format Automated Workflow User guide

For Research Use Only. Not for use in diagnostic procedures.

CarrierScan™ Assay 96-Array Format

Automated Workflow

USER GUIDE

for use with:

CarrierScan™ 1S 96F Array Plates

CarrierScan™ Reagent Kit 96 Reactions

Biomek™ FXP Target Prep Express Automated Workstation

Catalog Number 951951

Publication Number 703478

Revision 4

For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) will NOT BE LIABLE FOR SPECIAL, INCIDENTAL,

INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR

USE OF IT.

Important Licensing Information

This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited

Use Label Licenses.

Corporate entity

Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288

TRADEMARKS

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

Jitterbug is a trademark of Boekel Scientific. Microsoft, Excel, and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United

States and/or other countries. QIAGEN, CoralLoad, and REPLI-g are registered or registration-pending trademarks of the QIAGEN Group. Beckman Coulter, and

Biomek are either registered trademarks or trademarks of Beckman Coulter, Inc. TRobot is a trademark of Biometra GmbH. DNA Engine Tetrad, Bio-Rad,

Microseal, and Hard-Shell are registered trademarks of Bio-Rad Laboratories, Inc.

Manufacturer:

Affymetrix Pte Ltd

7 Gul Circle #2M-01

Keppel Logistics Building

Singapore 629563

Products:

CarrierScan™ 1S 96F Array Plate

Manufacturer:

Thermo Fisher Scientific Baltics UAB

V.A. Graiciuno 8, LT-02241

Vilnius, Lithuania

Products:

CarrierScan™ Reagent Kit 96 Reactions

Revision history: Pub. No. 703478

Revision Date Description

4 16 October 2020 Updated document to reflect new names for reagent kit components.

Added the plate option of Bio-Rad HSP9601 as an alternative for the

Bio-Rad HSP-9631 plate.

3 07 September 2018 Updated to reflect that Axiom™ Reference gDNA 103 has been removed

from the reagent kit and has been made available for purchase

separately.

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide 3

Contents

CHAPTER 1 The CarrierScan™ Assay 96-Array Format Automated

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP . . . . . . . . . . . . 11

What’s new . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Running multiple plate workflows. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

CHAPTER 2 Genomic DNA preparation and requirements. . . . . . . . . 15

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Sources of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

General requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Special requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Assessing the quality of genomic DNA using 1% Agarose E-gels . . . . . . . . . . . . . . . . . . 16

Genomic DNA extraction/purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Genomic DNA cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Thaw samples and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Quantitate and dilute test sample gDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Aliquot the diluted samples and the control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Freeze or proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Create a GeneTitan Array Plate Registration file. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

CHAPTER 3 Preparation before you start . . . . . . . . . . . . . . . . . . . . . . 23

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

CarrierScan™ arrays, reagents, and GeneTitan™ consumables required . . . . . . . . . . . . . . . 23

Reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Requirements and recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Room temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Special requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Contents

4CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide

Thermal cycler recommendations and protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

PCR plate type by thermal cycler for mPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Oven recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Plate centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Plate shakers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Equipment care and calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Seal, vortex, and centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

About the reagents and master mix preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Pipettes and pipetting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Matrix™ 25-mL reagent reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Equipment, consumables, and reagents required for mPCR preparation . . . . . . . . . . . . . . 32

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

CarrierScan™ Assay on the Biomek FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Before using the Biomek workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Breaking the light curtain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Pipette tip usage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Setting method preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Equipment, consumables, labware, and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Labware and materials used on the Biomek FXP workstation deck . . . . . . . . . . . . . . . . . . 43

Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Loading tray consumables onto the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . 58

Reagent block template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Reservoir stickers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Related Biomek FXP Target Prep Express documentation . . . . . . . . . . . . . . . . . . . . . . . . 62

CHAPTER 4 Multiplex PCR and Target preparation on the

Biomek FXP with Windows® 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

CarrierScan™ Assay on the Biomek FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Stage 1A: Multiplex PCR (mPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Input required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Equipment, consumables and reagents required. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

1: Prepare for mPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

2: Prepare the mPCR master mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

3: Set up the mPCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

4: Run the CarrierScan mPCR thermal cycler protocol and proceed. . . . . . . . . . . . . . . . . 68

Contents

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide 5

5: Store the mPCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Stage 1B: DNA amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . . . . . . . 70

1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

2. Thaw and prepare the reagents and Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

3. Run the DNA amplification step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Summary of DNA amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Stage 2: Fragmentation and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Input required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . . . . . . . 82

1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

2. Thaw and prepare the amplified DNA samples and reagents . . . . . . . . . . . . . . . . . . . . 84

3: mPCR spike-in to Amplification Plate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

4. Run the Fragmentation step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

4. Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Summary of Fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Stage 3: Centrifugation and drying pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Equipment and consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Stage 4: Resuspension, hybridization preparation, and QC . . . . . . . . . . . . . . . . . . . . . . . . . 96

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . . . . . . . 96

1. Preparing frozen pellets and Resuspension Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

2. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

3. Thaw and prepare the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

4. Run the Resuspension, hybridization preparation, and QC step . . . . . . . . . . . . . . . . . . 98

Summary of Resuspension and Hybridization Preparation . . . . . . . . . . . . . . . . . . . . . . . 105

Stage 5: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . 109

About Stage 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation . . 113

Equipment and consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

2. Prepare the reagents for GeneTitan Reagent Tray Preparation . . . . . . . . . . . . . . . . . . 116

3. Prepare the Sample Plate (if stored at –20°C) and the array plate . . . . . . . . . . . . . . . . 118

4. Prepare the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

5. Run the Preparation for GeneTitan™ step. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays . . . . . . . . . . . . 126

6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays. . . . . . . . . . . . . . . . 127

Contents

6CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide

6c. Complete Stage 4: Preparation for GeneTitan™ - Multiple Plate Workflow . . . . . . . . 127

Summary of Preparation for GeneTitan MC™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . 129

CHAPTER 5 Array processing with the GeneTitan™ MC Instrument. 134

Before using the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Email and telephone notifications from the GeneTitan™ MC Instrument . . . . . . . . . . . . . 139

GeneTitan™ MC Instrument lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Setup options for array plate processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Aborting a process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Stage 1: Create and upload GeneTitan Array Plate Registration file . . . . . . . . . . . . . . . . . . 144

Stage 2: Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Setup the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Load an array plate and hybridization tray onto the GeneTitan™ MC Instrument . . . . . . 150

Load a second array plate and hybridization tray onto the GeneTitan™ MC Instrument . 156

Status window prompts and actions required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Stage 3: Ligate, wash, stain, and scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Proper installation of GeneTitan™ consumables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Load trays onto the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Continuing the workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

Shutting down the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

CHAPTER 6 Processing 8 CarrierScan™ array plates per week . . . . 170

Overview of the eight-plate workflow of the CarrierScan™ Assay on the Biomek FXP . . . . . . . . 170

Duration of assay steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Thawing frozen plates of amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Thawing plates with frozen pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Simultaneous eight-plate workflow: Target preparation and array processing of the

CarrierScan™ Assay on the Biomek FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Day 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

Day 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

Day 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

Day 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Contents

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide 7

CHAPTER 7 Processing 3 CarrierScan™ array plates per week

using an overnight DNA Precipitation step . . . . . . . . . . . . . . . . . . . . . 195

Overview of the 3-plate workflow of the CarrierScan™ Assay on the Biomek FXP . . . . . . . 196

Duration of assay steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

Thawing frozen plates of amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Three-plate workflow: Target preparation and array processing of the CarrierScan™ Assay

on the Biomek FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Day 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

Day 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204

Day 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

Day 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Day 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

CHAPTER 8 Process 3 CarrierScan™ array plates per week using

a 3-hour DNA Precipitation step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

Overview of the 3-plate workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Duration of assay steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

Thawing frozen plates of amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Three-plate workflow: Target preparation and array processing . . . . . . . . . . . . . . . . . . . . 220

Day 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

Day 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Day 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

Day 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

CHAPTER 9 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Biomek FXP Target Prep Express . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

GeneTitan™ Multi-Channel Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Miscellaneous messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

Fluidic diagnostic messages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

Wash/Scan Resume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

Aborting a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

APPENDIX A Fragmentation quality control gel protocol . . . . . . . . . 239

Protocol for running a fragmentation quality control gel . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

E-Gels and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

Diluting the TrackIt™ Cyan/Orange Loading Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

Contents

8CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide

Fragmentation QC gel protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

APPENDIX B Sample quantitation after resuspension . . . . . . . . . . . 242

Protocol for sample quantitation after resuspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

Quantitate the diluted samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

Assess the OD readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

Suggested protocol for OD quantitation using the DTX 880 . . . . . . . . . . . . . . . . . . . . . . . . 244

If performing sample quantitation on a plate reader other than the DTX880 . . . . . . . . . . . 250

APPENDIX C Registering samples in GeneChip™

Command Console™. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

Creating a GeneTitan™ Array Plate Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

APPENDIX D Deionization procedure for GeneTitan™ trays

and covers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

Deionization procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

Ion-indicator cap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

APPENDIX E GeneTitan™ Multi-Channel Instrument Care . . . . . . . . 257

Cleaning and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Monthly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Every 6 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Servicing the outer enclosure fan filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Cleaning schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Cleaning procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Replacing the bottle filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

Removing and inspecting the filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

Replacing the filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

Replacing the xenon lamp in the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . 261

Lamp life/imaging device status notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

Removing the xenon lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

Replacing the lamp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

Resetting the lamp counter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

Log files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

GCC log files for GeneTitan™ MC Instrument Systems . . . . . . . . . . . . . . . . . . . . . . . . . . 266

Insufficient disk space notice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

Contents

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide 9

APPENDIX F mPCR quality control gel protocol. . . . . . . . . . . . . . . . 268

Protocol for running an mPCR quality control gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

E-Gels and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

Prepare NEB 50 bp DNA ladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

Preparing mPCR samples for gel analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

mPCR QC gel protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

APPENDIX G Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

General safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

10 CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide

1The CarrierScan™ Assay 96-Array

Format Automated Workflow

Overview

The Applied Biosystems™ CarrierScan™ Assay is an innovative, comprehensive, and

high-throughput microarray-based tool for the reliable and robust detection of

sequence and structural variation for preconception expanded carrier screening

research across a wide range of ethnicities. Developed in collaboration with experts

across the field of carrier screening research, the CarrierScan™ Assay is the industry’s

broadest content genetic analysis system specifically designed to provide detection of

both sequence and structural variants simultaneously, including biallelic and

multiallelic mutations such as single nucleotide variants (SNVs), insertion-deletion

variants (indels), as well as structural genomic variants, such as small intragenic

deletions and duplications (copy number variants).

CarrierScan Assay utilizes the same chemistry from the Axiom™ assay and GeneTitan™

Multi-Channel Instrument, a system that is preferred worldwide by genetic

researchers for the efficient workflow, high-throughput, economic pricing, and

reproducibility of results critical to multi-year data collection and analysis efforts.

Introduction

CarrierScan™ Assay 96-Array Format Automated Workflow is available as a bundled

kit that includes the arrays, reagents and consumables needed for processing one 96-

format plate. The automated protocol described in this user guide leverages the Axiom

2.0 Assay method on the Biomek FXP for target preparation and GeneTitan reagent

preparation. The multiplex PCR (mPCR) and mPCR spike-in steps are not part of the

automated method and must be executed off-deck, manually.

CarrierScan interrogates biallelic as well as multiallelic SNPs, indels and copy number

variation (CNV) in a single assay workflow. Starting with genomic DNA, the samples

are processed by performing an automated target preparation protocol followed by

automated processing of the array plates on the GeneTitan MC Instrument.

• Target preparation uses methods including DNA amplification, fragmentation,

purification and resuspension of the target in hybridization cocktail.

• The hyb-ready targets are then transferred to the Applied Biosystems GeneTitan™

Multi-Channel (MC) Instrument for automated, hands-free processing including

hybridization, staining, washing and imaging.

The CarrierScan Assay detects approximately 6,000 sequence and structural variants

in over 600 genes for the research of 600 diseases, informed by the American College

of Medical Genetics (ACMG) and the American College of Obstetricians and

Gynecologists (ACOG) guidelines from well-curated, prominent databases and peer-

reviewed literature (Grody, 2013; Landrum, 2016; Stenson, 2003; Zlotogora, 2015;

Langfelder-Schwind, 2014; Sosnay, 2013).

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide 11

Chapter 1 The CarrierScan™ Assay 96-Array Format Automated Workflow

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP1

The combination of these sequence and structural variants, in addition to CarrierScan’s

ability to precisely call variants in critical genes on a microarray, compliments Thermo

Fisher Scientific’s current solutions for carrier screening research using OpenArray™

Real-Time PCR, digital PCR, and Ion Ampliseq™ NGS panels.

CarrierScan Assay enables the assessment of more variants per sample in a single,

microarray-based, high-throughput, cost effective format. Complete with simple data

analysis and reporting software, this novel solution enables labs to quickly generate all

relevant carrier status data.

CarrierScan uses powerful biallelic and multiallelic detection, as well as state-of-the-

art copy number algorithms, and used in conjunction with curated annotations for

population frequencies, providing quick, reliable, and automated data analysis.

Array plates are processed on a GeneTitan™ MC Instrument controlled by Applied

Biosystems™ GeneChip™ Command Console™ 4.3 or higher. The resulting CEL files are

analyzed by Reproductive Health Research Analysis Software (RHAS) v 1.0 or higher,

a single source comprehensive software package for QC, genotyping, CN and carrier

status assessment.

For further information, please refer to "Related documentation" on page 274.

The CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP must

only be executed on controllers using the Windows 7 operating system.

CarrierScan™ Assay 96-Array Format Automated Workflow for

Biomek FXP

Running the CarrierScan Assay requires the following sets of steps:

1. Genomic DNA preparation—Resulting in samples that meet requirements

spelled out in Chapter 2, "Genomic DNA preparation and requirements" on

page 15.

2. A manual preparation of a multiplex PCR step (mPCR) followed by automated

target preparation of the samples (see Chapter 4, "Multiplex PCR and Target

preparation on the Biomek FXP with Windows® 7" on page 63).

3. Target preparation of the samples: Chapter 4, "Multiplex PCR and Target

preparation on the Biomek FXP with Windows® 7" on page 63.

4. Array processing, done with:

• GeneTitan MC Instrument

• GeneTitan Instrument Control software

• Applied Biosystems™ GeneChip™ Command Console Portal software

See Chapter 5, "Array processing with the GeneTitan™ MC Instrument" on

page 134.

A list of the required equipment and supplies for running the CarrierScan Assay using

the Biomek FXP for automated target preparation can be found in the CarrierScan™

Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub.

No. 703480.

Chapter 1 The CarrierScan™ Assay 96-Array Format Automated Workflow

What’s new

12 CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide

1

What’s new

This revision of the user guide introduces the option for a 3-hour DNA target

Precipitation step to enable a faster assay turnaround time, going from sample to CEL

file generation within 72 hours. The 3-hour Precipitation step shortens Stage 2:

Fragmentation and purification to enable the operator to advance to Stage 3: Centrifugation

and drying pellets, followed by Stage 4: Resuspension, hybridization preparation, and QC,

and the beginning of Stage 5: Preparation for the GeneTitan™ MC Instrument to initiate

hybridization on the GeneTitan MC instrument on day 2 of the assay workflow. See

Figure 1. Note that this workflow option requires approximately 9 hours to complete

these combined day 2 activities (fragmentation to initiation of hybridization on the

GeneTitan MC Instrument).

In addition, a recommended workflow is presented to support the processing of 3

plates per week using the shortened DNA Precipitation step. See Chapter 8 for details

regarding equipment and operator assumptions to support this workflow.

The standard CarrierScan Assay workflow, in which the DNA is precipitated

overnight, provides a convenient stopping point to support single operator assay

execution of 1 plate within an 8-hour workday. Recommended workflows are

presented to support the processing of 3 plates per week and 8 plates per week using

the standard overnight DNA Precipitation. See Chapter 6 and Chapter 7 for details

regarding equipment and operator assumptions to support these workflows.

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide 13

Chapter 1 The CarrierScan™ Assay 96-Array Format Automated Workflow

What’s new 1

Figure 1 CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP

10 μL

10 μL/well

20 μL/well

AMP Plate + mPCR

CarrierScan Assay overview – page 1 of 1

Stage 1A: mPCR

Duration: ~3 hours

Multiplex PCR

thermal cycler program

of mPCR PLATE

Stage 1B: DNA amplification

Duration: 23 hours

37°C incubation of

AMPLIFICATION PLATE

DAY 1 DAY 2

Stage 2:

Fragmentation and

purification

DAY 3

Duration: 16-24 hours

(or o ptional three-hour

precipitation step)

–20°C incubation of

PRECIPITATION PLATE

Stage 3:

Centrifugation and

drying pellets

Duration: 23.5-24 hours

Array Hybridization

DAY 4

Stage 5:

Preparation for the

GeneTitanTM MC

Instrument

Duration: ~ 12 hours

Fluidics: 4.5 hours

Scan: ~7.5 ho urs

mPCR spike-in

into AMP plate

Possible stopping point

mPCR Plate

gDNA plate setup

5 ng/μL of gDNA

Automated

Stage 1B: DNA

amplification

Stage 1A: mPCR

Manual

Stage 4:

Resuspension,

hybridization

preparation, and QC

Chapter 1 The CarrierScan™ Assay 96-Array Format Automated Workflow

What’s new

14 CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide

1

Running multiple

plate workflows

Thermo Fisher Scientific provides workflows that allow you to run a set of samples and

array plates through the protocol using a minimum of personnel and a forty-hour

week. The timing of steps is critical because of the following constraints:

• Incubation after DNA Amplification is 23 hours.

• Hybridization in the GeneTitan MC Instrument is 23.5 hours.

• Reagent trays for wash/stain/imaging must be prepared as Hybridization

finishes.

• Limits to when a second hybridization tray and array plate can be loaded into the

GeneTitan MC Instrument.

These limitations require careful timing. The details are covered in:

•Chapter 6, "Processing 8 CarrierScan™ array plates per week" on page 170.

•Chapter 7, "Processing 3 CarrierScan™ array plates per week using an overnight

DNA Precipitation step" on page 195.

•Chapter 8, "Process 3 CarrierScan™ array plates per week using a 3-hour DNA

Precipitation step" on page 214

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide 15

2Genomic DNA preparation and

requirements

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Sources of genomic DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

General requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Genomic DNA extraction/purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Genomic DNA cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Overview

The general requirements for genomic DNA (gDNA) sources and extraction methods

are described in this chapter. The success of this assay requires uniform amplification

of the genome starting with relatively intact gDNA. To achieve this, the gDNA must

be of high quality, and must be free of contaminants that may affect the enzymatic

reactions to be performed.

For this protocol, you use the CarrierScan™ Reagent Kit 96 Reactions (Cat. No. 931933).

Reference Genomic DNA 103 (Cat. No. 951957) is available for purchase separately.

This DNA meets the requirements outlined below, and is used as a control. The size

and purity of sample gDNA can be compared with those of the control DNA to assess

sample quality. The control DNA should also be used routinely as an experimental

positive control and for troubleshooting purposes.

Assay performance can vary for gDNA samples that do not meet the general

requirements described below. However, the reliability of any given result should be

assessed in the context of overall experimental design and goals.

Sources of genomic DNA

The following sources of human gDNA have been successfully tested in the

CarrierScan Assay 96-Array Format Automated Workflow for Biomek FXP with DNA

that meets the above requirements.

• Whole blood

• Buccal cell

• Saliva

• Cell lines

Other sample types have not been verified in this assay and are not currently

supported.

Note: DNA derived from formalin-fixed paraffin-embedded (FFPE) blocks should not

be used with this assay.

Chapter 2 Genomic DNA preparation and requirements

General requirements

16 CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide

2

General requirements

• Starting DNA must be double-stranded for the purpose of accurate concentration

determination.

• DNA must be of high purity.

DNA should be free of DNA polymerase inhibitors. Examples of inhibitors

include high concentrations of heme (from blood) and high concentrations of

chelating agents (i.e., EDTA). The gDNA extraction/ purification method should

render DNA that is generally salt-free because high concentrations of particular

salts can also inhibit enzyme reactions. DNA purity is indicated by OD260/OD280

and OD260/OD230 ratios. The OD260/OD280 ratio should be between 1.8 and 2.0 and

the OD260/OD230 ratio should be greater than 1.5. We recommend that DNA

samples that do not meet these criteria be cleaned up as described under

"Genomic DNA cleanup" on page 18.

• DNA must not be degraded.

The approximate average size of gDNA may be assessed on a 1% agarose gel

using an appropriate size standard control. Approximately 90% of the DNA must

be greater than 10 Kb in size. Control DNA can be run on the same gel for side-

by-side comparison.

• For the CarrierScan Assay, it is recommended that the assay plate contain a

minimum of 48 female samples to ensure accurate genotyping and copy number

performance. For questions regarding this recommendation contact Thermo

Fisher Scientific Technical Support.

Special

requirements

Preamplification area

Precautions are required when manipulating genomic DNA to avoid contamination

with foreign DNA amplified in other reactions and procedures. It is recommended that

genomic DNA manipulations are performed in a dedicated preamplification room or

area separate from the main laboratory.

This preamplification area should have a dedicated set of pipettes and plasticware. If

no dedicated area is available, use of a dedicated bench or a dedicated biosafety hood

and dedicated pipettes is suggested. If no dedicated bench or biosafety hood is

available, a set of dedicated pipettes is recommended.

Assessing the

quality of genomic

DNA using 1%

Agarose E-gels

We recommend this quality control step to assess the quality of the gDNA prior to

starting the assay.

Equipment and reagents recommended

Table 1 E-Gel® and reagents required

Item Source

Mother E-Base Device EB-M03

Daughter E-Base Device EB-D03

E-Gel® 48 1% agarose gels G8008-01

RediLoad™750026

E-Gel® 96 High Range DNA Marker 12352-019

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide 17

Chapter 2 Genomic DNA preparation and requirements

General requirements 2

Guidelines for preparing the Genomic DNA Plate for gel analysis

• Loading a DNA mass of 10 ng to 20 ng per well is recommended. If lower amounts

are loaded, omission of the loading dye is recommended in order to improve

visualization. Loading 25 ng gDNA per well can improve the image.

• Add 3 µL of 0.1X of RediLoad dye to each sample.

• Bring each sample to a total volume of 20 µL using H2O (for example, if the

volume of genomic DNA is 5 µL, add 3 µL of RediLoad, and bring to 20 µL total

by adding 12 µL of H2O).

• Seal, vortex, then centrifuge.

Run a 48-lane 1% agarose e-gel

1. Power on for E-Base (red light).

2. Push the Power/Prg button to make sure the program is at EG mode (not EP).

3. Insert the two 48 well 1% agarose e-gels into the slots.

4. Remove 2 combs.

5. Load 20 µL from the above plate onto two 48 well 1% agarose e-gels.

6. Load 15 µL of diluted High Range DNA Marker (1:3 dilution or ~0.34 X from

stock) into all marker wells (as needed).

7. Fill all empty wells with water.

8. Adjust the run time to ~27 minutes

9. Push the Power/Prg button again (it changes from red to green).

When run time is reached (the ladder band reaches the end of the lane), the system

automatically shuts off. The gel is then ready for imaging.

Figure 2 shows gel images of intact gDNA (that is suitable for use in the CarrierScan™

Assay) and degraded gDNA samples. Customers whose gDNA is degraded (similar to

the image in Figure 2) should perform a test experiment to investigate the performance

of their samples in the CarrierScan Assay prior to beginning any large scale genotyping

projects.

Figure 2 Gel images showing intact gDNA and degraded gDNA.

Chapter 2 Genomic DNA preparation and requirements

Genomic DNA extraction/purification methods

18 CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide

2

Genomic DNA extraction/purification methods

Genomic DNA extraction and purification methods that meet the general

requirements outlined above should yield successful results. Methods that include

boiling or strong denaturants are not acceptable because the DNA would be rendered

single-stranded and can no longer be accurately quantitated using a PicoGreen-based

assay.

Genomic DNA cleanup

If a gDNA preparation is suspected to contain inhibitors, the following cleanup

procedure can be used:

1. Add 0.5 volumes of 7.5 M NH4OAc, 2.5 volumes of absolute ethanol (stored at

–20°C), to gDNA.

2. Vortex and incubate at –20°C for 1 hour.

3. Centrifuge at 12,000 x g in a microcentrifuge at room temperature for 20 minutes

4. Remove supernatant and wash pellet with 80% ethanol.

5. Centrifuge at 12,000 x g at room temperature for 5 minutes

6. Remove the 80% ethanol and repeat the 80% ethanol wash 1 more time.

7. Resuspend the pellet in reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0,

0.1 mM EDTA).

(See the CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP

Site Preparation Guide, Pub. No. 703480 for reagents, equipment, labware and

consumables for the CarrierScan Assay).

Genomic DNA preparation

This step must be done before proceeding with the mPCR and DNA amplification

stages. The genomic DNA (gDNA) you process using the CarrierScan Assay should

meet the general requirements listed earlier in this chapter. The amount of gDNA is

50 ng for the mPCR step and 100 ng for the CarrierScan whole-genome amplification

step.

To prepare gDNA:

"Thaw samples and control"

"Quantitate and dilute test sample gDNA"

"Aliquot the diluted samples and the control"

"Freeze or proceed"

"Create a GeneTitan Array Plate Registration file"

Table 2 Input requirements for CarrierScan Assay 96-Array Format Automated Workflow for Biomek FXP

Assay step Volume per well Input mass per well gDNA concentration

Stage 1A: mPCR 10 µL 50 ng 5 ng/µL

Stage 1B: DNA Amplification 20 µL 100 ng 5 ng/µL

CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide 19

Chapter 2 Genomic DNA preparation and requirements

Genomic DNA preparation 2

Duration Thirty minutes to 1 hour for reagents to thaw, and 30 minutes for setup.

Equipment,

consumables, and

reagents required

Equipment and consumables

The equipment and consumables listed in Table 3 are required for this stage.

Reagents

The reagents listed in Table 4 are required for this stage.

Table 3 Equipment and consumables required for genomic DNA preparation

Quantity Item

As required Adhesive seals for plates

1 Ice bucket, filled with ice

1 each Pipettes:

• Single channel P10 or P20 • Optional: Multichannel P10 or P20

As required Pipette tips

1 Plate, deepwell: Beckman Deepwell Titer, polypropylene; Cat. No.

267007

1 96 well PCR plate (Bio-Rad HSS-9641 for Applied Biosystems 9700,

Applied Biosystems Veriti™, Applied Biosystems ProFlex™, and Bio-Rad

HSP-9631 or HSP-9601 for Eppendorf Master™ Cycler pro S)

1 Plate centrifuge

1 Plate spectrophotometer (required only if no OD measurements

available for samples)

1 Vortexer

Table 4 Reagents required for genomic DNA preparation

Reagent Source

Reference Genomic DNA 103 (use as a positive control, –20°C) 951957

User-supplied

Reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) 75793

Quant-iT™ PicoGreen™ dsDNA Assay Kit P7589

Chapter 2 Genomic DNA preparation and requirements

Genomic DNA preparation

20 CarrierScan™ Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) User Guide

2

Thaw samples and

control

Thaw the components listed below to room temperature:

• gDNA samples

• Reference Genomic DNA 103

To thaw, either:

• Place items on benchtop for 1 hour

• Thaw in a water bath:

a. Fill a small plastic dish with ultra-pure water (such as Millipore water). Do

not overfill as the level of the water should not overflow when the control

DNA tubes or plates are placed in the bath.

b. Thaw the sealed Sample Plate and control DNA tubes for a 30 minutes.

c. Remove the Sample Plate and/or control DNA tube from the water bath and

wipe-dry using lab wipes. Ensure the outside is completely dry before

opening the Sample Plate or tube to minimize any contamination, which can

lead to reaction failure.

Quantitate and

dilute test sample

gDNA

Quantitate and dilute test sample gDNA

1. Gently vortex (50% maximum) and centrifuge the gDNA and Reference Genomic

DNA 103.

2. Recommendation: quantitate each sample (e.g., using the Quant-iT™ PicoGreen®

dsDNA Kit).

3. Using reduced EDTA TE buffer, dilute each sample to a concentration of 5 ng/µL.

4. Seal, vortex and centrifuge.

Note: Do not dilute the Reference Genomic DNA 103 control. It is already at a working

concentration.

Note: We strongly recommend that you determine your sample concentrations using

the Quant-iT PicoGreen assay (Cat. No. P7589). Sample concentration determined by

UV absorbance is often inaccurate and can yield different results.

Aliquot the diluted

samples and the

control

Next, the samples and control are placed in a deepwell plate for DNA amplification

and into a 96-well PCR plate for mPCR.

Note: Make sure gDNA is well mixed before plating.

Amplification sample plate

• Beckman Deepwell Titer Plate; Cat. No. 267007

Aliquot diluted samples and the Reference Genomic DNA 103 control to the deepwell

plate

1. Aliquot 20 µL of each diluted gDNA sample to the Beckman Deepwell Titer Plate

as shown in Figure 3.

2. Pipet 20 µL of control DNA. We recommend including at least 1 positive control

on each plate.

3. Seal and centrifuge.

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Thermo Fisher Scientific CarrierScan Assay 96-Array Format Automated Workflow User guide

Thermo Fisher Scientific CarrierScan Assay 96-Array Format Automated Workflow User guide

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